Project Summary: Microglia are the innate immune cells of the CNS that mediate opposing deleterious pro- inflammatory and protective anti-inflammatory functions in Alzheimer's Disease (AD). Specific inhibitors of pro- inflammatory microglial functions with high CNS bioavailability are desperately needed as disease-modifying treatments for AD are lacking. Kv1.3 is a microglial potassium channel that regulates membrane potential and pro-inflammatory functions and is highly expressed by amyloid beta plaque-associated microglia in human AD brains. While ShK peptides (ShK-186, 192, ShK-F6CA) are the most potent and selective inhibitors of Kv1.3 channels, their disease-modifying effects in AD are limited by poor CNS penetrance across the blood brain barrier (BBB). We propose to engineer an apolipoprotein E3 (apoE3)-based nanoparticle capable of BBB- crossing delivery of ShK-F6CA, a fluorescein-conjugated peptide that selectively blocks Kv1.3 potassium channels on activated microglia in neuroinflammation, and to examine its delivery efficacy using both an in vitro BBB model of neuroinflammation and an in vivo mouse model of AD. We have successfully engineered the proposed nanoparticle platforms that mimic apoE3-based high-density lipoprotein (HDL-E3), a natural nanoparticle known to traverse the CNS via receptor-mediated transport across the BBB. With our recent progress in the incorporation of a fluorescent-conjugated ShK analog (ShK-F6CA) into the engineered HDL- based nanoparticle (eHNP), we hypothesize that eHNP-E3-ShK-F6CA can cross the BBB to inhibit pro- inflammatory microglial functions in a mouse model of AD pathology. Our high-throughput microfluidic technology and biomimetic microsystems engineering approach will contribute to high-throughput production and screening of eHNPs. We expect that our bioinspired CNS delivery strategy of Kv1.3 channel blocker will enable microglial Kv1.3 channel blockade and attenuate the neuroinflammation in an in vitro BBB model and an in vivo transgenic mouse model of rapidly progressing AD pathology (5xFAD). With recent successful completion of ShK-based therapeutics (ShK-186) in the human Phase 1 and 2A clinical trials for systemic autoimmune disorders, our approach using bioinspired nanotechnology engineered to enable BBB-crossing transport of a microglial ion channel blocker will attenuate amyloid-associated neuroinflammation and create the possibility of altering the course of AD. The successful outcomes will serve as a foundation for translating the basic research and technology to the clinics, with particular pertinence to accelerating advanced CNS delivery of therapeutic and diagnostic agents.